The crosslinking of hemoglobin to erythrocyte structural proteins and the oxidation of membrane lipids are thought to bc important features of oxidative pathways that lead to erythrocyte lysis and/or removal. The crosslinking and peroxidative processes are triggered by the reactions of hemoglobin with H202 and other redox active agents. Analogous myoglobin-dependent crosslinking and peroxidative reactions may be involved in reperfusion damage to myocardial tissue. The purpose of this project is to define the chemical mechanisms of the interactions of hemoglobin and myoglobin with peroxides and oxidant drugs and to explore the relationship of these reactions to hemolysis and tissue damage. The immediate goals are to define the amino acid residues involved in hemoglobin and myoglobin crosslinking, to determine the nature of the H202-dependent crosslink between the protein and the heme group in hemoglobin and myoglobin, to prepare site specific myoglobin mutants and to use them to define the roles of tyrosine and cysteine residues in deleterious processes, to identify the meso-heme adducts formed in reactions of the globins with aryl- and alkylhydrazines and other carbon radical precursors, and to elucidate the mechanisms by which the hemoproteins catalyze the oxidation of unsaturated lipids. Of particular interest in the latter context is the protein mediated cooxidation mechanism suggested by work carried out in the previous period of support. The results should help clarify the chemical mechanisms of oxidant-dependent hemolysis and reperfusion injury and should provide information relevant to the mechanisms of other physiologically important processes mediated by protein radicals.